The Prognostic Value of CD39 as a Marker of Tumor-Specific T Cells in Triple-Negative Breast Cancer in Asian Women.

Triple-negative breast cancer (TNBC) has a poor prognosis with limited therapeutic options available for affected patients. Efforts are ongoing to identify surrogate markers for tumor-specific CD8+ T cells that can predict the response to immune checkpoint inhibitor (ICI) therapies, such as programmed cell death protein 1 or programmed cell death ligand-1 blockade. We have previously identified tumor-specific CD39+CD8+ T cells in non-small cell lung cancer that might help predict patient responses to programmed cell death protein 1 or programmed cell death ligand-1 blockade. Based on this finding, we conducted a comparative interrogation of TNBC in an Asian cohort to evaluate the potential of CD39 as a surrogate marker of tumor-specific CD8+ T cells. Using ICI-treated TNBC mouse models (n = 24), flow cytometric analyses of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes revealed that >99% of tumor-specific CD8+ T cells also expressed CD39. To investigate the relationship between CD39+CD8+ T-cell density and CD39 expression with disease prognosis, we performed multiplex immunohistochemistry staining on treatment-naive human TNBC tissues (n = 315). We saw that the proportion of CD39+CD8+ T cells in human TNBC tumors correlated with improved overall survival, as did the densities of other CD39+ immune cell infiltrates, such as CD39+CD68+ macrophages. Finally, increased CD39 expression on CD8+ T cells was also found to predict the response to ICI therapy (pembrolizumab) in a separate cohort of 11 TNBC patients. These findings support the potential of CD39+CD8+ T-cell density as a prognostic factor in Asian TNBC patients.

Platelet TLR7 is essential for the formation of platelet-neutrophil complexes and low-density neutrophils in lupus nephritis.

OBJECTIVES: Platelets and low-density neutrophils (LDNs) are major players in the immunopathogenesis of SLE. Despite evidence showing the importance of platelet-neutrophil complexes (PNCs) in inflammation, little is known about the relationship between LDNs and platelets in SLE. We sought to characterize the role of LDNs and Toll-like receptor 7 (TLR7) in clinical disease. METHODS: Flow cytometry was used to immunophenotype LDNs from SLE patients and controls. The association of LDNs with organ damage was investigated in a cohort of 290 SLE patients. TLR7 mRNA expression was assessed in LDNs and high-density neutrophils (HDNs) using publicly available mRNA sequencing datasets and our own cohort using RT-PCR. The role of TLR7 in platelet binding was evaluated in platelet-HDN mixing studies using TLR7-deficient mice and Klinefelter syndrome patients. RESULTS: SLE patients with active disease have more LDNs, which are heterogeneous and more immature in patients with evidence of kidney dysfunction. LDNs are platelet bound, in contrast to HDNs. LDNs settle in the peripheral blood mononuclear cell (PBMC) layer due to the increased buoyancy and neutrophil degranulation from platelet binding. Mixing studies demonstrated that this PNC formation was dependent on platelet-TLR7 and that the association results in increased NETosis. The neutrophil:platelet ratio is a useful clinical correlate for LDNs, and a higher NPR is associated with past and current flares of LN. CONCLUSIONS: LDNs sediment in the upper PBMC fraction due to PNC formation, which is dependent on the expression of TLR7 in platelets. Collectively, our results reveal a novel TLR7-dependent crosstalk between platelets and neutrophils that may be an important therapeutic opportunity for LN.

Integrative multiomics enhancer activity profiling identifies therapeutic vulnerabilities in cholangiocarcinoma of different etiologies.

OBJECTIVES: Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. DESIGN: Integrative multiomics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining and single-cell spatial transcriptomics were used to explore the immunogenicity of diverse CCA. RESULTS: We identified three distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumour growth in in vitro and in vivo models. The first group (ESTRO), with mostly fluke-positive CCAs, displayed activation in estrogen signalling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive to oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intratumour differences in AA mutation load were correlated to intratumour variation of different immune cell populations. CONCLUSION: Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumourigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits.

Hypoxia-regulated carbonic anhydrase IX (CAIX) protein is an independent prognostic indicator in triple negative breast cancer.

BACKGROUND: The effect of extracellular microenvironment (hypoxia and pH) has been regarded as a key hallmark in cancer progression. The study aims to investigate the effects of carbonic anhydrase IX (CAIX), a key hypoxia-inducible marker, in triple-negative breast cancer (TNBC) in correlation with clinicopathological parameters and predicting survival outcomes. METHODS: A total of 323 TNBC cases diagnosed at the Department of Anatomical Pathology, Singapore General Hospital from 2003 to 2013 were used. Immunohistochemical staining (IHC) was performed using CAIX antibody and digital mRNA quantification was performed using NanoString assays. CAIX membranous expression was correlated with clinicopathological parameters using Chi-squared test or Fisher's exact tests. Disease-free survival (DFS) and overall-survival (OS) were estimated using Kaplan-Meier analysis and compared between groups with the log-rank test. RESULTS: Forty percent of TNBCs were observed to express CAIX protein and demonstrated significant association with larger tumour size (P = 0.002), higher histological grade (P < 0.001), and significantly worse disease-free survival (DFS) and overall survival (OS) (after adjustment: HR = 2.99, 95% CI = 1.78-5.02, P < 0.001 and HR = 2.56, 95% CI = 1.41-4.65, P = 0.002, respectively). Gene ontology enrichment analysis revealed six significantly enriched cellular functions (secretion, cellular component disassembly, regulation of protein complex assembly, glycolytic process, cellular macromolecular complex assembly, positive regulation of cellular component biogenesis) associated with genes differentially expressed (CAIX, SETX, WAS, HK2, DDIT4, TUBA4α, ARL1). Three genes (WAS, SETX and DDIT4) were related to DNA repair, indicating that DNA stability may be influenced by hypoxia in TNBC. CONCLUSIONS: Our results demonstrate that CAIX appears to be a significant hypoxia-inducible molecular marker and increased CAIX protein levels are independently associated with poor survival in TNBC. Identification of CAIX-linked seven gene-signature and its relationship with enriched cellular functions further support the implication and influence of hypoxia-mediated CAIX expression in TNBC tumour microenvironment.

Quantitative stain-free imaging and digital profiling of collagen structure reveal diverse survival of triple negative breast cancer patients.

BACKGROUND: Stromal and collagen biology has a significant impact on tumorigenesis and metastasis. Collagen is a major structural extracellular matrix component in breast cancer, but its role in cancer progression is the subject of historical debate. Collagen may represent a protective layer that prevents cancer cell migration, while increased stromal collagen has been demonstrated to facilitate breast cancer metastasis. METHODS: Stromal remodeling is characterized by collagen fiber restructuring and realignment in stromal and tumoral areas. The patients in our study were diagnosed with triple-negative breast cancer in Singapore General Hospital from 2003 to 2015. We designed novel image processing and quantification pipelines to profile collagen structures using numerical imaging parameters. Our solution differentiated the collagen into two distinct modes: aggregated thick collagen (ATC) and dispersed thin collagen (DTC). RESULTS: Extracted parameters were significantly associated with bigger tumor size and DCIS association. Of numerical parameters, ATC collagen fiber density (CFD) and DTC collagen fiber length (CFL) were of significant prognostic value for disease-free survival and overall survival for the TNBC patient cohort. Using these two parameters, we built a predictive model to stratify the patients into four groups. CONCLUSIONS: Our study provides a novel insight for the quantitation of collagen in the tumor microenvironment and will help predict clinical outcomes for TNBC patients. The identified collagen parameters, ATC CFD and DTC CFL, represent a new direction for clinical prognosis and precision medicine. We also compared our result with benign samples and DICS samples to get novel insight about the TNBC heterogeneity. The improved understanding of collagen compartment of TNBC may provide insights into novel targets for better patient stratification and treatment.

Tertiary lymphoid structures and associated plasma cells play an important role in the biology of triple-negative breast cancers.

PURPOSE: Triple-negative breast cancers (TNBC) are aggressive tumours that exhibit abundant lymphoid infiltrates which modulate tumour behaviour. Recent findings suggest that TNBC with higher densities of plasma cells are associated with a favourable prognosis, and tertiary lymphoid structures (TLS) have prognostic significance. Here, we studied the phenotype and function of plasma cells in TNBCs by assessing their association with IgG Kappa light chain expression, B cells, and TLS. METHODS: A retrospective analysis of 269 TNBC cases was performed. Tumour-infiltrating CD38+ plasma cells, CD20+ B cells, and TLS were evaluated on conventional haematoxylin-eosin-stained and immunohistochemical-stained sections of TNBC. We then selected TNBC cases demonstrating the highest and lowest densities of plasma cells, and examined their association with TLS, B cells, as well as immunoglobulin expression using Opal-Vectra multiplex immunofluorescence (IF). RESULTS: TNBC with high density of plasma cells showed significantly higher numbers of IgG Kappa+ CD38+ cells (p = 0.0089, p < 0.0001), and higher numbers of TLS (p < 0.0001), compared to TNBC with low density of plasma cells. TNBC with high density of plasma cells also showed higher numbers of CD20+ B cells in the tumour core (p < 0.0001), invasive margin (p < 0.0001), as well as stromal (p = 0.015) compartments. CONCLUSION: TNBC with high density of plasma cells are associated with higher numbers of IgG Kappa+ CD38+ cells, CD20+ B cells, and TLS. Further studies to characterize the function of plasma cell infiltrates and how they may interact with other tumour-infiltrating lymphocytes and TLS in TNBC may help improve existing immunotherapy strategies.

Evaluation of phospho-histone H3 in Asian triple-negative breast cancer using multiplex immunofluorescence.

PURPOSE: We used multiplex immunofluorescence (mIF) to determine whether mitotic rate represents an independent prognostic marker in triple-negative breast cancer (TNBC). Secondary aims were to confirm the prognostic significance of immune cells in TNBC, and to investigate the relationship between immune cells and proliferating tumour cells. METHODS: A retrospective Asian cohort of 298 patients with TNBC diagnosed from 2003 to 2015 at the Singapore General Hospital was used in the present study. Formalin-fixed, paraffin-embedded breast cancer samples were analysed on tissue microarrays using mIF, which combined phospho-histone H3 (pHH3) expression with cytokeratin (CK) and leukocyte common antigen (CD45) expression to identify tumour and immune cells, respectively. RESULTS: Multivariate analysis showed that a high pHH3 index was associated with significantly improved overall survival (OS; p = 0.004), but this was not significantly associated with disease-free survival (DFS; p = 0.22). Similarly, multivariate analysis also revealed that a pHH3 positive count of > 1 cell per high-power field in the malignant epithelial compartment was an independent favourable prognostic marker for OS (p = 0.033) but not for DFS (p = 0.250). Furthermore, a high CD45 index was an independent favourable prognostic marker for DFS (p = 0.018), and there was a significant positive correlation between CD45 and pHH3 index (Spearman rank correlation coefficient, 0.250; p < 0.001). CONCLUSIONS: Mitotic rates as determined by pHH3 expression in epithelial cells are significantly associated with improved survival in TNBC. mIF analysis of pHH3 in combination with CK and CD45 could help clinicians in prognosticating patients with TNBC.

Behaviour and characteristics of low-grade ductal carcinoma in situ of the breast: literature review and single-centre retrospective series.

AIMS: Ductal carcinoma in situ (DCIS) of the breast is a heterogeneous disease that has risen to prominence and more recently controversy, with the advent of screening mammography. Debate concerning the true biological potential of low nuclear grade DCIS continues to challenge therapeutic considerations. In this study, we carried out a comprehensive literature review of the behaviour, outcomes and current management trials of low-grade DCIS, as well as a retrospective study of a large single institutional series of low-grade DCIS diagnosed at our hospital. METHODS AND RESULTS: The study cohort comprised 195 cases of low-grade DCIS diagnosed at the Singapore General Hospital from 1994 to 2010. Clinicopathological parameters and follow-up data were retrieved and compared between screen-detected and symptomatic low-grade DCIS. Immunohistochemistry was performed for ER, PR and HER2. Among 195 cases, 123 (63.1%) were screen-detected, while 72 (36.9%) were symptomatic. Screen-detected cases had frequent calcifications (P < 0.001) and were smaller (P = 0.018) than symptomatic cases. All cases were ER-positive and rate of PR expression was high. No HER2 overexpression was observed. Mean and median follow-up periods were 107.8 and 109.6 months, respectively. Six patients recurred ipsilaterally, and one patient developed direct distant metastasis. One breast cancer-related death was recorded. Positive surgical margins (P = 0.023) were significantly associated with a higher risk of ipsilateral recurrences, as well as poorer disease-free survivals (P = 0.010). CONCLUSION: Our data indicate that low-grade DCIS may be followed by invasive recurrences and even metastatic disease, requiring more study before being regarded as innocuous and indolent.

An integrated automated multispectral imaging technique that simultaneously detects and quantitates viral RNA and immune cell protein markers in fixed sections from Epstein-Barr virus-related tumours.

BACKGROUND/AIM: Epstein-Barr virus (EBV) is an oncovirus that is commonly associated with the development of lymphomas and epithelial carcinomas. In the era of immunotherapy, histological evaluation of EBV-related cancers is currently a multi-sample, multi-technique process requiring separate time-consuming detection of EBV-encoded small RNAs by in situ hybridisation (ISH), and parallel labelling of sections for cancer-associated protein markers. METHODS: Using EBV-associated tumours as proof-of-concept for feasibility, here we developed an approach that allows simultaneous detection of EBV RNAs and multiple protein markers such as PD-L1, EBV-LMP, CD8, CD4, CD20, CD30 and CD15on a single tissue section based on our recently reported automated staining protocol. RESULTS: We successfully combined multiplex immunofluorescence (mIF) to detect 3 abovementioned protein markers involved in cancer, with ISH, and applied the protocol to f tissue samples from patients diagnosed with EBV-associated pulmonary lymphoepithelioma-like carcinoma (LELC), gastric carcinoma and Hodgkin's Lymphoma. Empowered by the Vectra 3 Automated Quantitative Pathology Imaging System, we demonstrate the utility and potential of this integrated approach to concurrently detect and quantitate viral RNA and protein biomarkers of immune and tumour cells. CONCLUSION: This study represents an important step forward in the research and diagnosis of EBV-associated cancers, and could be readily modified to include other proteins and RNA markers to apply to other malignancies. More importantly, the novel automated ISH-mIF protocol that we detailly described here could also be readily reproduced by most of the diagnostic and research lab to future projects that aim to look at both RNA and protein markers.

Higher densities of Foxp3+ regulatory T cells are associated with better prognosis in triple-negative breast cancer.

PURPOSE: The role of Forkhead Box Protein 3 (Foxp3) expressing regulatory T cells (Tregs) in breast cancer remains unclear. We examined the abundance and localisation of total T cells, B cells and Tregs within samples from triple-negative breast cancers (TNBCs) and asked whether these parameters were associated with clinicopathological features of the cancer or clinical outcomes. METHODS: Samples from TNBCs diagnosed between 2003 and 2010 in Singapore were divided into "high" and "low" intra-tumoural or stromal groups, based on whether they had higher or lower than median densities of specific tumour-infiltrating lymphocyte populations (CD3+ total T cells, Foxp3+CD3+ Tregs, or CD20+ B cells) in the intra-tumoural space or stroma. RESULTS: Of the 164 samples, patients bearing tumours with high Tregs within their intra-tumoural, but not stromal, areas experienced significantly longer overall and disease-free survival compared to individuals with low Treg densities. These "high intra-tumoural Treg" tumours were also characterised by relatively higher frequencies of CD8+ T cells and CD20+ B cells, and expressed significantly higher levels of some genes associated with inflammation, immune cell functions and trafficking, altogether consistent with a more "immune-activated" tumour microenvironment, in contrast to tumours bearing lower densities of Tregs. CONCLUSIONS: In summary, the combination of high densities of intra-tumoural Tregs, CD8+ T cells and CD20+ B cells represents a favourable prognostic panel in TNBCs. These data also indicate new avenues for further investigation on the interaction between immune cell types within the tumour microenvironment and highlight the potential of Treg density and localisation within tumours to affect clinical outcome.

A five-gene reverse transcription-PCR assay for pre-operative classification of breast fibroepithelial lesions.

BACKGROUND: Breast fibroepithelial lesions are biphasic tumors and include fibroadenomas and phyllodes tumors. Preoperative distinction between fibroadenomas and phyllodes tumors is pivotal to clinical management. Fibroadenomas are clinically benign while phyllodes tumors are more unpredictable in biological behavior, with potential for recurrence. Differentiating the tumors may be challenging when they have overlapping clinical and histological features especially on core biopsies. Current molecular and immunohistochemical techniques have a limited role in the diagnosis of breast fibroepithelial lesions. We aimed to develop a practical molecular test to aid in distinguishing fibroadenomas from phyllodes tumors in the pre-operative setting. METHODS: We profiled the transcriptome of a training set of 48 formalin-fixed, paraffin-embedded fibroadenomas and phyllodes tumors and further designed 43 quantitative polymerase chain reaction (qPCR) assays to verify differentially expressed genes. Using machine learning to build predictive regression models, we selected a five-gene transcript set (ABCA8, APOD, CCL19, FN1, and PRAME) to discriminate between fibroadenomas and phyllodes tumors. We validated our assay in an independent cohort of 230 core biopsies obtained pre-operatively. RESULTS: Overall, the assay accurately classified 92.6 % of the samples (AUC = 0.948, 95 % CI 0.913-0.983, p = 2.51E-19), with a sensitivity of 82.9 % and specificity of 94.7 %. CONCLUSIONS: We provide a robust assay for classifying breast fibroepithelial lesions into fibroadenomas and phyllodes tumors, which could be a valuable tool in assisting pathologists in differential diagnosis of breast fibroepithelial lesions.

Role of epithelial-mesenchymal transition markers in triple-negative breast cancer.

Triple-negative breast cancers (TNBCs) are a heterogeneous group of breast tumours that are often associated with adverse pathological characteristics, poorer clinical outcomes and lack of targeted therapeutic options. Epithelial-mesenchymal transition, which plays a crucial role in tumour development and progression, is characterised by a transition from epithelial to mesenchymal phenotype and loss of proteins involved in maintaining cell junctions. We aimed to correlate protein expression of E-cadherin, Snail2 and transforming growth factor beta (TGF-ß) with clinicopathological parameters and survivals of a series of patients with TNBC. The study cohort comprised 767 TNBCs diagnosed at the Department of Pathology, Singapore General Hospital from 1994 to 2012. Immunohistochemistry was performed on sections cut from tissue microarrays using the polymeric method. Staining intensity and percentage of positive tumour cells were evaluated and correlated with clinicopathological findings and clinical outcomes. Loss of E-cadherin expression, Snail2 positivity, cytoplasmic and nuclear expression of TGF-ß were observed in 265 (35.2 %), 241 (32.0 %), 272 (36.2 %) and 262 (34.8 %) tumours, respectively. Histological grade significantly correlated with Snail2 positivity (P < 0.001) and loss of membranous E-cadherin expression (P = 0.003). Nuclear expression of TGF-ß was inversely correlated with histological grade (P = 0.010). Median follow-up was 73 months, with a maximum of 236 months. Despite a graphical curve for earlier recurrence in patients with tumours harbouring a combinational phenotype of loss of membranous E-cadherin and positive Snail2 expression, there was no statistical significance. Similarly for women with tumours expressing cytoplasmic TGF-ß, graphical representation showed poorer metastasis-free survival but without statistical significance. Loss of membranous E-cadherin and positive Snail2 expression are significantly associated with high-grade TNBCs. More work is needed to improve understanding of the role of TGF-ß in TNBC.

Prognostic significance of epithelial-mesenchymal transition proteins Twist and Foxc2 in phyllodes tumours of the breast.

Epithelial-mesenchymal transition (EMT), an important process during embryonic development, is reportedly exploited during tumour progression. Deregulation of EMT-related molecules has been shown in many malignancies, including breast carcinoma. We aim to investigate the clinical relevance and prognostic significance of EMT proteins, Twist and Foxc2, in breast phyllodes tumours (PTs). The study cohort comprised 271 PTs diagnosed from 2003 to 2010. Of these, 188 (69.4 %) were benign, 60 (22.1 %) borderline, and 23 (8.5 %) malignant. Immunohistochemistry for Twist and Foxc2 was performed on tissue microarray sections. Percentage of tumour cells stained was evaluated and correlated with clinicopathological parameters and clinical outcome. Twist and Foxc2 stromal nuclear expression was associated with tumour grade (P = 0.038 and 0.012). Foxc2 stromal nuclear expression was positively correlated with epithelial expression (P < 0.001), tumour relapse, and metastasis (P = 0.037). Furthermore, stromal nuclear immunoreactivity of Twist and Foxc2 was interrelated (P < 0.001). Tumours expressing Foxc2 and those co-expressing both Twist and Foxc2 revealed a shorter time to recurrence (P < 0.001 and 0.001) and death (P = 0.044 and 0.015). Twist and Foxc2 stromal expression in PTs was significantly correlated with tumour grade and worse histological features. In addition, expression of Foxc2 and co-expression of Twist and Foxc2 in the stroma of PTs contributed to poorer prognosis. Clinical relevance of EMT-related molecules may be worthy of further investigation in PTs.

Characteristics and behaviour of screen-detected ductal carcinoma in situ of the breast: comparison with symptomatic patients.

Breast cancer is the most common malignancy in Singapore women. Ductal carcinoma in situ (DCIS) is the putative, non-obligate precursor of the majority of invasive breast cancers. The efficacy of the Singapore breast-screening pilot project in detecting early stage breast cancer led to the launch of a national breast-screening programme, BreastScreen Singapore (BSS), in January 2002. In this study, we compared clinicopathological and immunohistochemical characteristics, as well as clinical outcomes, between screen-detected and symptomatic DCIS. The study cohort comprised 1202 cases of DCIS diagnosed at Singapore General Hospital from 1994 to 2010. Comparison of clinicopathological parameters, immunohistochemical results of ER, PR, HER2, CK14, EGFR, and 34ßE12, and clinical outcomes was carried out between the 2 groups. Amongst 1202 cases, 610 (50.7%) were screen-detected and 592 (49.3%) were symptomatic DCIS. Screen-detected cases were smaller in size (P < 0.001), of lower nuclear grade (P = 0.004), and more frequently expressed ER (P < 0.001). Luminal A phenotype was more frequently observed in screen-detected DCIS, while triple-negative and HER2 phenotypes were more common in symptomatic DCIS (P < 0.001). The basal-like phenotype was also more frequent in symptomatic DCIS (P = 0.041). Mean and median follow-up was 99.7 and 97.8 months, respectively, with a maximum follow-up of 246.0 months. More symptomatic patients developed invasive recurrences compared to screen-detected patients (P = 0.001). A trend for better disease-free survival was observed in screen-detected patients (P = 0.076). Patients who were screen-detected experienced better overall survival than those with symptomatic DCIS (P = 0.007). Our data indicate a more favourable outcome of screen-detected DCIS patients confirming the role of BSS in early identification of this curable disease.

Novel genetic aberrations in breast phyllodes tumours: comparison between prognostically distinct groups.

Phyllodes tumours of the breast are uncommon fibroepithelial neoplasms which pose management challenges due to difficulties in accurate prediction of clinical behaviour, as histological assessment has its limitations. Molecular studies have improved the understanding of these rare tumours but such findings are scant. We aimed to investigate genetic aberrations in phyllodes tumours stratified according to clinical behaviour, to identify potential genes contributing to disease progression. Twenty phyllodes tumours were separated into prognostically distinct categories depending on whether they had recurred/metastasized within the follow-up period. DNA extracted from FFPE materials was subjected to Affymetrix OncoScan™ FFPE Express molecular inversion probe microarray platform for analysis of copy number changes and mutational status. Results were cross validated with Sanger sequencing, FISH and immunohistochemistry. A higher number of chromosomal aberrations were observed in cases which recurred/metastasized, with median events of 19 compared to 3.5 in cases which did not recur/metastasize. High-level amplification and homozygous deletions were detected exclusively in the former group. Regions of high-level amplification included MDM4 (1q32.1), RAF1 (3p25), EGFR (7p12) and PDZD2 (5p13.3). EGFR amplification was confirmed on FISH and accompanied by intense EGFR immunostaining. Regions of homozygous deletion included CDKN2A (9p21) and MACROD2 (20p12.1). Homozygous deletion of 9p21 which involved CDKN2A was accompanied by loss of protein expression. No mutations were identified in all samples. These findings provide insights into identifying target genes and pathways exploited by phyllodes tumours, which would aid future development of individualised therapy.

Application of Artificial Intelligence to In Vitro Tumor Modeling and Characterization of the Tumor Microenvironment.

In vitro tumor models have played vital roles in enhancing the understanding of the cellular and molecular composition of tumors, as well as their biochemical and biophysical characteristics. Advances in technology have enabled the evolution of tumor models from two-dimensional cell cultures to three-dimensional printed tumor models with increased levels of complexity and diverse output parameters. With the increase in complexity, the new generation of models is able to replicate the architecture and heterogeneity of the tumor microenvironment more realistically than their predecessors. In recent years, artificial intelligence (AI) has been used extensively in healthcare and research, and AI-based tools have also been applied to the precise development of tumor models. The incorporation of AI facilitates the use of high-throughput systems for real-time monitoring of tumorigenesis and biophysical tumor properties, raising the possibility of using AI alongside tumor modeling for personalized medicine. Here, the integration of AI tools within tumor modeling is reviewed, including microfluidic devices and cancer-on-chip models.

Spatial transcriptomics reveal topological immune landscapes of Asian head and neck angiosarcoma.

Angiosarcomas are rare malignant tumors of the endothelium, arising commonly from the head and neck region (AS-HN) and recently associated with ultraviolet (UV) exposure and human herpesvirus-7 infection. We examined 81 cases of angiosarcomas, including 47 cases of AS-HN, integrating information from whole genome sequencing, gene expression profiling and spatial transcriptomics (10X Visium). In the AS-HN cohort, we observed recurrent somatic mutations in CSMD3 (18%), LRP1B (18%), MUC16 (18%), POT1 (16%) and TP53 (16%). UV-positive AS-HN harbored significantly higher tumor mutation burden than UV-negative cases (p = 0.0294). NanoString profiling identified three clusters with distinct tumor inflammation signature scores (p < 0.001). Spatial transcriptomics revealed topological profiles of the tumor microenvironment, identifying dominant but tumor-excluded inflammatory signals in immune-hot cases and immune foci even in otherwise immune-cold cases. In conclusion, spatial transcriptomics reveal the tumor immune landscape of angiosarcoma, and in combination with multi-omic information, may improve implementation of treatment strategies.

Immunohistochemical scoring of LAG-3 in conjunction with CD8 in the tumor microenvironment predicts response to immunotherapy in hepatocellular carcinoma.

Introduction: Immune checkpoint blockade (ICB) is a systemic therapeutic option for advanced hepatocellular carcinoma (HCC). However, low patient response rates necessitate the development of robust predictive biomarkers that identify individuals who will benefit from ICB. A 4-gene inflammatory signature, comprising CD8, PD-L1, LAG-3, and STAT1, was recently shown to be associated with a better overall response to ICB in various cancer types. Here, we examined whether tissue protein expression of CD8, PD-L1, LAG-3, and STAT1 predicts response to ICB in HCC. Methods: HCC samples from 191 Asian patients, comprising resection specimens from 124 patients (ICB-naïve) and pre-treatment specimens from 67 advanced HCC patients treated with ICB (ICB-treated), were analyzed for CD8, PD-L1, LAG-3, and STAT1 tissue expression using multiplex immunohistochemistry followed by statistical and survival analyses. Results: Immunohistochemical and survival analyses of ICB-naïve samples showed that high LAG-3 expression was associated with shorter median progression-free survival (mPFS) and overall survival (mOS). Analysis of ICB-treated samples revealed that high proportions of LAG-3+ and LAG-3+CD8+ cells pre-treatment were most closely associated with longer mPFS and mOS. Using a log-likelihood model, adding the total LAG-3+ cell proportion to the total CD8+ cell proportion significantly increased the predictive values for mPFS and mOS, compared with the total CD8+ cell proportion alone. Moreover, levels of CD8 and STAT1, but not PD-L1, were significantly correlated with better responses to ICB. After analyzing viral-related and non-viral HCC samples separately, only the LAG3+CD8+ cell proportion was significantly associated with responses to ICB regardless of viral status. Conclusion: Immunohistochemical scoring of pre-treatment levels of LAG-3 and CD8 in the tumor microenvironment may help predict ICB benefits in HCC patients. Furthermore, immunohistochemistry-based techniques offer the advantage of being readily translatable in the clinical setting.